Week 3 - Electrophoresis, Chromatography, Mass Spectrometry

What is chromatography?

A method in which components of a mixture are separated based on their differential interactions with two chemical or physical phases.

What is the mobile phase in chromatography?

The phase that travels through the system and carries the sample components once the sample has been applied or injected.

What is the stationary phase in chromatography?

The phase that is held within the system by a support.

How are components separated in chromatography?

Components with the strongest interactions with the stationary phase are retained more and move through the system more slowly; components with weaker interactions move faster.

What is a chromatograph?

An instrument used in chromatography that provides a response related to the amount of a compound eluting from a column as a function of elution time or volume of mobile phase.

What is a chromatogram?

The resulting plot of the detector response versus time or volume.

What is retention time (tR)?

The average time required for a chemical to pass through the column.

What is retention volume (VR)?

The average volume of mobile phase required for a chemical to pass through the column.

What is void time (tM)?

The elution time for a compound that has no interaction with the stationary phase and is not retained.

What is void volume (VM)?

The elution volume for a compound that has no interaction with the stationary phase and is not retained.

What is column chromatography?

A type of chromatographic system that uses a column (or tube) to contain the stationary phase and support.

What is planar chromatography?

A type of chromatography where the support and stationary phase are present on a plane or open surface; used primarily for qualitative analysis.

How are chromatographic methods classified based on the mobile phase?

Gas chromatography (GC) uses a gas mobile phase; liquid chromatography (LC) uses a liquid mobile phase.

What is gas-solid chromatography (GSC)?

A type of chromatography classified by the stationary phase, where the same material acts as both the stationary phase and the support.

What is the procedure for planar chromatography?

The stationary phase is coated onto a flat surface; the sample is added as a small spot or band; the support is placed in an enclosed container with one edge in contact with the mobile phase; the mobile phase travels across the plane by capillary action.

What is the retention factor (Rf) in planar chromatography?

Rf = distance solute (Ds) / distance solvent (Df).

What does an Rf value of 0.67 mean?

The solute traveled 67% of the distance that the solvent traveled.

How are chemicals identified in planar chromatography?

By comparing their Rf values with those of reference compounds examined on the same stationary phase, support, and mobile phase.

How are separated components detected in planar chromatography?

By their natural colour, response to UV light, fluorescence, or visualization with reagents that form coloured products.

What is one clinical application of planar chromatography?

Analysis of amniotic fluid to determine lecithin-to-sphingomyelin ratios.

What is liquid chromatography (LC)?

A type of chromatography in which the mobile phase is a liquid; based on the distribution of chemicals between a liquid mobile phase and a stationary phase.

What is the dominant type of chromatography currently used in clinical laboratories?

Liquid chromatography (LC).

What is adsorption chromatography?

Also known as liquid-solid chromatography; retention is based on competition between the sample and the mobile phase for adsorptive sites on the solid stationary phase.

What are the three types of adsorbents used in adsorption chromatography?

1. Polar acidic (adsorbs polar compounds, works well with basic substances), 2. Polar basic (adsorbs polar compounds, works well with acidic substances), 3. Nonpolar (charcoal, polystyrene).

Why is adsorption chromatography not widely used in clinical laboratories?

Because of technical problems with the preparation of the stationary phase.

What is partition chromatography?

Also called liquid-liquid chromatography; separation is based on relative solubility in a nonpolar solvent and an aqueous solvent.

What is the support material in most types of partition chromatography?

Silica.

What is ion-exchange chromatography?

A type of LC in which ions are separated by their adsorption onto a support that contains fixed charges at its surface.

What determines whether cations or anions bind to the column in ion-exchange chromatography?

The charge of the groups that make up the stationary phase.

What is size-exclusion chromatography?

An LC technique that separates analytes based on size using a porous support with an inert surface and a range of pore sizes.

In size-exclusion chromatography, which components elute first?

Larger components elute first because they can enter only a few or none of the pores.

What is affinity chromatography?

An LC method that uses biologically related interactions for the retention and separation of chemicals (e.g., antibody-antigen binding or enzyme-substrate interactions).

What is HPLC?

High-Performance Liquid Chromatography; uses pressure for fast separations, controlled temperature, in-line detectors, and gradient elution techniques.

What are the parts of an HPLC system?

Pump, column, sample injectors, detectors, and recorders.

What is the purpose of the pump in HPLC?

Uses force to push the mobile phase through the column.

What types of pumps are used in HPLC?

Pneumatic, syringe, reciprocating, or hydraulic amplifier pumps.

What is the most common material used for the column in HPLC?

Silica gel.

What is the purpose of the detector in HPLC?

Monitors the eluate as it leaves the column and produces an electronic signal proportional to the concentration of each separated component.

What types of detectors are used in HPLC?

Spectrophotometers or photodiode arrays (usually connected to a mass spectrometer).

What is the purpose of the recorder in HPLC?

Records detector signal versus the time the mobile phase passed through the instrument.

How is retention time used in HPLC?

To identify compounds when compared with standard retention times run under identical conditions.

What is peak area proportional to in HPLC?

The concentration of the compounds that produced the peaks.

What is gas chromatography (GC)?

A type of chromatography where the mobile phase is a gas; used to separate and analyze compounds that are naturally volatile or can be converted into a volatile form.

What is the mobile phase typically in gas chromatography?

An inert gas such as nitrogen or helium.

What are separations in gas chromatography based on?

Differences in the vapour pressures of the injected compounds and in the interactions of these compounds with the stationary phase.

What is gas-solid chromatography?

A type of GC where the same material acts as both the stationary phase and the support; chemicals are retained by adsorption to the surface of the support (inorganic material like silica or alumina).

What affects retention in gas-solid chromatography?

Surface area, size of the pores, and the types of functional groups present.

What is gas-liquid chromatography (GLC)?

A type of GC where the stationary phase is a liquid placed as a coating or layer on the support.

What is column bleed?

The process where some of the liquid stationary phase leaves the column over time, resulting in a change in the ability to retain chemicals.

How is column bleed minimized?

By using a bonded phase instead of a liquid.

What is electrophoresis?

The separation of a mixture of electro-active colloidal particles in solution under the influence of an electrical field.

Is electrophoresis a separation method or a measurement technique?

It is a separation method, NOT a measurement technique.

What are the routine uses of electrophoresis?

Separation of serum proteins, glycoproteins, lipoproteins, amino acids, iso-enzymes, and immunoglobulins.

Why is an alkaline solution used in electrophoresis?

Body pH is slightly alkaline, so using an alkaline solution gives the proteins a negative charge.

What is the basic principle of electrophoresis?

When a voltage is applied across an electrolyte solution, charged particles migrate toward the positive anode (if negative) or the negative cathode (if positive).

What does the electrophoretic mobility of a particle depend on?

Its net charge and ionic radius.

What are the four basic parts of an electrophoresis system?

1. Electroactive particles, 2. Electrolyte solution (buffer), 3. Physical support or stabilizing medium, 4. High voltage DC power supply (usually 60V for ~1hr).

What is the result of electrophoresis?

The sample is separated into bands or "zones," which are visualized and then quantitated by densitometry.

What are the four steps of the electrophoresis procedure?

1. Place sample on support medium soaked in buffer, 2. Place support medium and sample in electrophoretic chamber and apply current, 3. Visualize using a staining method, 4. Quantitate using a densitometer.

What is the purpose of the support medium?

It provides the matrix in which separation takes place.

What are three types of support media used in electrophoresis?

Cellulose acetate, agarose, and polyacrylamide.

What is agarose made from?

Agar; used for DNA electrophoresis.

What is polyacrylamide used for?

Serum proteins; should be purchased because acrylamide is a neurotoxin and carcinogen.

What are the two roles of buffers in electrophoresis?

1. Carries the applied current, 2. Establishes the pH at which electrophoresis is performed.

What pH is used for electrophoresis and why?

pH = 8.6 (alkaline) to keep proteins negatively charged.

What does the buffer determine in electrophoresis?

The type of electrical charge on the solute, the extent of ionization of the solute, and the electrode toward which the solute will migrate.

What are four factors affected by buffer concentration?

1. Conductance of the support, 2. Thickness of the ionic cloud surrounding the charged molecule, 3. Rate of molecule's migration, 4. Sharpness of the electrophoretic zones.

What happens when buffer concentration is increased?

It yields sharper band separations but produces more heat due to increased current levels, which can cause denaturation of heat-labile proteins.

What happens to the ionic cloud when buffer strength is too high?

The ionic cloud increases in size, and the molecule becomes hindered in its movement (endosmosis).

What is endosmosis (electroendosmosis)?

The movement of buffer ions and solvent relative to the fixed support.

Why does cellulose acetate become negatively charged during electrophoresis?

Because of adsorption of hydroxyl ions.

What causes positive ions to move toward the cathode in endosmosis?

The application of current.

What happens to protein migration when the ion cloud is too strong?

Proteins (which are negatively charged) normally move to the positive side, but if the ion cloud is too strong, they won't migrate to the positive side and could move backwards to the negative side.

What stain is used for proteins in electrophoresis?

Amido Black B, Coomassie Brilliant Blue, Ponceau S.

What stain is used for isoenzymes in electrophoresis?

Nitrotetrazolium blue.

What stain is used for lipoproteins in electrophoresis?

Fat Red, Oil Red O, Sudan Black.

What stain is used for DNA fragments in electrophoresis?

Ethidium Bromide, SYBR safe green.

What stain is used for CSF proteins in electrophoresis?

Silver Nitrate, Methylene Blue.

What is a densitometer?

An instrument used to quantify stained zones by measuring the absorbance of each fraction as the gel is moved past a photometric optical system.

What does a densitometer display?

An electropherogram on a computer display.

What does the software convert each peak into?

Percent total or absolute concentration from the total protein.

What is required to calculate the concentration of each fraction?

The total protein value.

What are the five fractions of serum protein electrophoresis?

Albumin, alpha-1, alpha-2, beta, and gamma.

What are the advantages of capillary electrophoresis?

Ability to apply much higher voltage and ease of automation.

What type of tube is used in classic capillary electrophoresis?

A small-bore fused silica capillary tube.

What is isoelectric focusing?

A technique that separates amphoteric compounds, such as proteins, with increased resolution in a medium possessing a stable pH gradient.

What happens to a protein during isoelectric focusing?

The protein becomes "focused" at a point on the gel as it migrates to a zone where the pH of the gel matches the protein's pI.

What is the isoelectric point (pI)?

The pH at which the charge of the protein becomes zero and its migration ceases.

What is mass spectrometry useful for?

Identifying and determining elemental composition and structure of compounds, as well as performing quantitative analysis of components of samples.

What is a mass spectrometer?

An analytical instrument that ionizes molecules and then separates and measures the mass-to-charge ratio (m/z) of the ionized molecules and their fragments.

What happens to the sample in a mass spectrometer?

It is first volatilized (must be gaseous) and then ionized.

What is a mass spectrum?

A plot of the relative abundance of ions as a function of their m/z ratio.

When an ion has a single charge (z=1), what does the m/z ratio equal?

The mass of the ion.

What is the molecular ion?

The unfragmented ion.

What are fragment ions?

Ions formed when a molecular ion breaks into smaller pieces.

What is the base peak?

The ion with the highest abundance in the mass spectrum, assigned a relative value of 100%.

What are the five components of all mass spectrometers?

1. Ion source, 2. Vacuum system, 3. Mass analyzer, 4. Detector, 5. Computer.

Why must the instrumentation process occur in a vacuum?

To prevent molecules from colliding into each other.

What is electron ionization?

A gas-phase molecule is bombarded by electrons emitted from a heated filament; electrons eject an electron from the analyte molecule, producing a radical cation.

In electron ionization, what is the mass spectral pattern dominated by?

Fragment ions; can be matched to entries in a mass spectral library.